Agrobacterium is a genus of gram-negative soil bacteria which cause neoplastic cell growth in dicotyledonous plants by means of the natural system they possess for transferring phytohormone-producing DNA to plant cells. The T-(transferred)region is a segment of the large so-called Ti-(tumour inducing)plasmid (200 kb) harbored by all virulent Agrobacterium strains. (Occasionally, the T-region is also referred to as T-DNA.) Mobilization of the T-region to the plant cells is mediated by another region of the Ti-plasmid, which is called the vir-(virulence)region, in a process that resembles bacterial conjugation (see for reviews: Melchers LB and Hooykaas PJJ (1987) In: Miflin BJ (ed) Oxford surveys of plant and cell biology, vol 4. Oxford Univ Press, London/New York, pp 167-220; Zambryski P (1988) Annu. Rev. Genet. 22:1-30; Zambryski P, Tempe J, Schell J (1989) Cell 56:193-201).
This natural gene transfer system can be used to co-transfer DNA not naturally present in the T-region. The T-region is flanked by two 24 bp imperfect direct repeats (T-DNA borders) which are the only cis-essential elements for the transfer process; i.e. the borders must be linked to the DNA to be transferred to the plant cell. An ideal vector system for transferring DNA to plant cells should comprise the DNA to be transferred into the plant cell flanked on both sides by T-DNA borders, although it has been established that the right T-DNA border, preferably in conjunction with a region just outside the T-region and referred to as "overdrive", suffices for the transfer of DNA. Although not essential, the presence of this overdrive increases the efficiency of transfer of the DNA flanking the right border.
The second essential element, the vir region, can be provided in trans, i.e. not physically linked to the T-region. This finding has originated in the development of a binary vector system, in which the T-region, comprising DNA to be co-transferred to the plant cell, is cloned into a broad-host-range plasmid which is capable of replication in Agrobacterium and E. coli, while the vir functions are supplied by a disarmed helper plasmid which does not contain a T-region (Hoekema A, Hirsch PR, Hooykaas PJJ, Schilperoort RA (1983) Nature 310:115-120; European Patent EP-B 120 516); Bevan M (1984) Nucl. Acids. Res. 22:8711-8721). A binary vector system wherein the T-region is located on the chromosome of the Agrobacterium strain has also been disclosed (EP-B 176 112).
Although a binary vector system is very versatile, broad-host-range plasmids are less suitable as routine cloning vectors; they are large and generally have a low copy number. Moreover, they are usually unstable in Agrobacterium in the absence of selective pressure.
As an alternative to a binary vector system, one can use a so-called cointegrate system. Typically, the vir-functions reside on a Ti-plasmid and the DNA to be co-transferred has to be recombined into this Ti-plasmid. All cloning steps can be done using an E. coli vector, whereafter this vector is introduced into Agrobacterium, for instance by triparental mating or electroporation. Subsequently, homologous recombination between the vector and the acceptor Ti-plasmid has to take place, via a single or double cross-over, employing large regions of homology between the E. coli vector and the acceptor Ti-plasmid. The regions of homology can for instance be engineered into the acceptor Ti-plasmid by replacing the DNA originally comprised between the T-DNA borders on said plasmid with cloning vector sequences, such as pBR322 (Van Haute E, Joos H, Maes M, Warren G, Van Montagu M, Schell J. (1983) EMBO J. 2:411-418; Zambryski P, Joos H, Genetello C, Leemans J, Van Montagu M, Schell J. (1983) EMBO J 12:2143-2150; Deblaere R, Bytebier B, De Greve H, deboeck F, Schell J, Van Montagu M, Leemans J (1985) Nucl Acids Res 13:4777-4788). It is also possible to employ helper plasmids which are free of T-DNA borders as acceptor plasmids; in this case it will be necessary to provide at least the right T-DNA border operably linked to the DNA to be introduced into a plant cell on the cloning vector. A disadvantage of this system is that homologous recombination, especially the favor double crossing over, is not a very efficient and precise process in Agrobacterium strains, necessitating tedious selection of the cells harboring the desired cointegrates, before they can be used in plant transformation. An additional disadvantage of the resulting cointegrate plasmids resides in the fact that large regions of repetitive sequences occur, which may cause instability.
It is possible to perform the recombination steps in E. coli instead of Agrobacterium, as described in EP-B 120 515, and subsequently transferring the cointegrate plasmid to Agrobacterium for use in plant transformation.
It would be advantageous if recombination can be performed in Agrobacterium, without the need for large regions of homology between the acceptor plasmid and the cloning vector.